Beruflich Dokumente
Kultur Dokumente
IMMUNOMODULATORY EFFECTS Stefan Raduner, Adriana Majewska, Jian-Zhong Chen, Xiang-Qun Xie, Jacques Hamon, Bernard Faller, Karl-Heinz Altmann and Jrg Gertsch
J. Biol. Chem. 2006, 281:14192-14206. doi: 10.1074/jbc.M601074200 originally published online March 17, 2006
Access the most updated version of this article at doi: 10.1074/jbc.M601074200 Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites. Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts
Supplemental material:
http://www.jbc.org/content/suppl/2006/03/22/M601074200.DC1.html This article cites 65 references, 24 of which can be accessed free at http://www.jbc.org/content/281/20/14192.full.html#ref-list-1
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 20, pp. 1419214206, May 19, 2006 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Received for publication, February 3, 2006, and in revised form, March 13, 2006 Published, JBC Papers in Press, March 17, 2006, DOI 10.1074/jbc.M601074200
Stefan Raduner, Adriana Majewska, Jian-Zhong Chen, Xiang-Qun Xie, Jacques Hamon, Bernard Faller, Karl-Heinz Altmann, and Ju rg Gertsch1 From the Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, CH-8093 Zu rich, Switzerland, the Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, Texas 77204-5037, and Novartis Institutes for BioMedical Research, Discovery Technologies, 4002 Basel, Switzerland
Alkylamides (alkamides) from Echinacea modulate tumor necrosis factor mRNA expression in human monocytes/macrophages via the cannabinoid type 2 (CB2) receptor (Gertsch, J., Schoop, R., Kuenzle, U., and Suter, A. (2004) FEBS Lett. 577, 563569). Here we show that the alkylamides dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (A1) and dodeca-2E,4E-dienoic acid isobutylamide (A2) bind to the CB2 receptor more strongly than the endogenous cannabinoids. The Ki values of A1 and A2 (CB2 60 nM; CB1 >1500 nM) were determined by displacement of the synthetic high affinity cannabinoid ligand [3H]CP-55,940. Molecular modeling suggests that alkylamides bind in the solvent-accessible cavity in CB2, directed by H-bonding and - interactions. In a screen with 49 other pharmacologically relevant receptors, it could be shown that A1 and A2 specifically bind to CB2 and CB1. A1 and A2 elevated total intracellular Ca2 in CB2-positive but not in CB2-negative promyelocytic HL60 cells, an effect that was inhibited by the CB2 antagonist SR144528. At 50 nM, A1, A2, and the endogenous cannabinoid anandamide (CB2 Ki >200 nM) up-regulated constitutive interleukin (IL)-6 expression in human whole blood in a seemingly CB2dependent manner. A1, A2, anandamide, the CB2 antagonist SR144528 (Ki <10 nM), and also the non-CB2-binding alkylamide undeca-2E-ene,8,10-diynoic acid isobutylamide all significantly inhibited lipopolysaccharide-induced tumor necrosis factor , IL-1, and IL-12p70 expression (5500 nM) in a CB2-independent manner. Alkylamides and anandamide also showed weak differential effects on anti-CD3- versus anti-CD28-stimulated cytokine expression in human whole blood. Overall, alkylamides, anandamide, and SR144528 potently inhibited lipopolysaccharide-induced inflammation in human whole blood and exerted modulatory effects on cytokine expression, but these effects are not exclusively related to CB2 binding. the treatment of the common cold and upper respiratory infections are contradictory (4, 5). In contrast to the significant investments into the clinical assessment of the efficacy of Echinacea (6), the molecular mechanism of action of this medicinal plant has remained elusive, and comprehensive studies on the immunomodulatory actions of Echinacea constituents are scarce. We have reported previously that unsaturated fatty acid N-alkylamides (alkylamides) from Echinacea preparations can modulate the expression of TNF-2 in human monocytes and macrophages (Ms) in vitro (7). We ascribed these effects to interactions of alkylamides with the cannabinoid type 2 receptor (CB2) on monocytes, resulting in the activation of c-Jun N-terminal kinase, mitogen-activated protein kinase, and of the nuclear factor B (NF-B), which ultimately leads to TNF- mRNA expression (7). In the same study it was demonstrated that alkylamides inhibit LPS-stimulated TNF- protein expression from isolated monocytes/Ms. These findings were independently confirmed in a more recent study, which demonstrated binding of alkylamides from E. angustifolia to rodent cannabinoid receptors and inhibition of fatty acid amide hydrolase, the enzyme that controls the half-life of the endogenous cannabinoid ligand anandamide (8). Cannabinoid receptors belong to the G-protein-coupled receptor (GPCR) family and are ubiquitously found in the central nervous system and in the periphery. So far, two types of receptors have been characterized, which are referred to as type 1 (CB1) and type 2 (CB2) receptors. The CB1 receptor is predominantly but not exclusively found in the nervous system, whereas the CB2 receptor is mainly expressed on immune cells and in the spleen (9, 10). Because of its role in the cellular immune system, the CB2 receptor is of particular interest for our ongoing studies on the immunomodulatory activity of alkylamides from Echinacea. In fact, the CB2 receptor is abundantly expressed in different types of inflammatory and immune-competent cells (11, 12), and there is accumulating evidence that the CB2 receptor plays a role in inflammatory reactions and the immune response (1315), as well as related pathophysiological conditions (16, 17). It is also well established that cannabinoids mediate both inhibitory and stimulatory effects on the immune system by modulating cytokine expression (18, 19). These differential effects are also dependent on
2
Purple coneflower (Echinacea purpurea and Echinacea angustifolia) preparations are widely used herbal medicines for the treatment of the common cold and upper respiratory infections (1, 2). It is generally believed that Echinacea affords its benefits through interactions with the immune system (3), but data on the clinical efficacy of Echinacea in
* The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2. 1 To whom correspondence should be addressed. Tel.: 41-44-633-7374; Fax: 41-44-6331366; E-mail: juerg.gertsch@pharma.ethz.ch.
The abbreviations used are: TNF-, tumor necrosis factor ; 2-AG, 2-arachidonoyl-glycerol; CB, cannabinoid receptor; FACS, fluorescence-activated cell sorting; GM-CSF, granulocyte colony-stimulating factor; GPCR, G-protein-coupled receptor; LPS, lipopolysaccharide; NF-B, nuclear factor B; IL, interleukin; Ms, macrophages; PLC, phospholipase C; PMA, phorbol ester (12-tetradecanoylphorbol-13 acetate); BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; A1, dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide; A2, dodeca-2E,4E-dienoic acid isobutylamide; A3, undeca-2E-ene,8,10diynoic acid isobutylamide; CBA, cytometric bead array.
14192 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 NUMBER 20 MAY 19, 2006 Downloaded from http://www.jbc.org/ by guest on September 19, 2013
EXPERIMENTAL PROCEDURES
Cell CultureHuman promyelocytic leukemia non-CB2-expressing (negative) HL60 cells (obtained from Prof. Dr. Verena Dirsch, Vienna, Austria) were grown in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum, 1 g/ml fungizone (amphotericin B), 100 units/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine (all from Invitrogen). Human promyelocytic leukemia CB2-expressing (positive) HL60 cells (obtained from the ATCC, CCL-240) were grown in Iscoves modified Dulbeccos medium with 4 mM L-glutamine and 1.5 g/liter sodium bicarbonate (ATCC, Manassas, VA) supplemented with 20% fetal bovine serum, 1 g/ml fungizone (amphotericin B), 100 units/ml penicillin, and 100 g/ml streptomycin. The human CB2-expressing CHO-K1 cells were grown in the same medium as the CB2negative HL60 cells but supplemented with 400 g/ml G418 (10131027; Invitrogen). All cells were grown in a humidified incubator at 37 C and 5% CO2. Human Peripheral Whole Blood Cultures10 ml of peripheral whole blood was obtained from healthy volunteers in the early afternoon by a
MAY 19, 2006 VOLUME 281 NUMBER 20 JOURNAL Downloaded from http://www.jbc.org/ by guest on September 19, 2013 OF BIOLOGICAL CHEMISTRY
14193
14194 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 NUMBER 20 MAY 19, 2006 Downloaded from http://www.jbc.org/ by guest on September 19, 2013
FIGURE 2. Cellular CB2 expression and [3H]CP-55,940 displacement from human CB2 receptor. A, expression of the CB2 receptor on HEK293 cells (RBXCB2M; PerkinElmer Life Sciences), CB2-transfected CHO-K1 cells, and CB2-positive promyelocytic HL60 cells (CCL-240; ATCC) as determined by semiquantitative Western blotting and FACS. B, Hill plot showing displacement of [3H]CP-55,940 by anandamide. C, displacement of [3H]CP-55,940 by A1 showing a biphasic curve (n 4 S.D.). D, low concentration part of displacement curve by A1 (n 4 S.D.).
FACSCanto) with the CBA Analysis Software; BD Biosciences). The results were expressed as pg/ml and then analyzed for their relative expression (control versus treated sample). The lower limit of detection for each cytokine was determined as 20 pg/ml. Drugs and AntibodiesDodeca-2E,4E-dienoic acid isobutylamide (A2) was isolated from E. purpurea as published previously (31) for E. angustifolia root material. Dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (A1) and undeca-2E-en-8,10-diynoic acid isobutylamide (A3) were gifts from R. Lehmann (MediHerb, Australia). Compounds were checked for identity and integrity by thin layer chromatography and 1H NMR (500 MHz Bruker) spectroscopy prior to use. Anandamide, 2-AG, AM630, and CP-55,940 were obtained from Tocris Cookson Ltd. (UK). SR144528 was obtained as a gift from Sanofi-Synthe labo Recherche (France). Fluo3/AM, Pluronic F-127, and the monoclonal anti-rabbit fluorescein isothiocyanate antibody were purchased from Sigma. CB2 rabbit polyclonal antibody (3561) was obtained from Abcam (UK) and was tested for differential binding to immune cells. The radioligand [3H]CP-55,940 was obtained from PerkinElmer Life Sci-
ences. Anandamide, LPS (E. coli, serotype 055:B5), and PMA (from Euphorbiaceae) were obtained from Fluka Chemie, Switzerland. Thapsigargin was purchased from Alexis Biochemicals, Switzerland. Monoclonal CD3 (555336) and CD28 (348040) were purchased from Pharmingen. Calculations and StatisticsResults are expressed as mean values S.D. or S.E. for each examined group. Statistical significance of differences between groups was determined by the Students t test (paired t test) with GraphPad Prism software. Outliners in a series of identical experiments were determined by Grubbs test (ESD method) with set to 0.05. Statistical differences between treated and vehicle control groups were determined by Students t test for dependent samples. Differences between the analyzed samples were considered as significant if p 0.05.
RESULTS
The solubility of the lipophilic alkylamides A1 and A2 is limited in aqueous solutions as detected by a Tyndall effect at concentrations
MAY 19, 2006 VOLUME 281 NUMBER 20 JOURNAL Downloaded from http://www.jbc.org/ by guest on September 19, 2013 OF BIOLOGICAL CHEMISTRY
14195
Ki of human CB2
nM
Ki of human CB1 6210 800 1940 370 40,000 40.7 2a 37 472b 40,000 NDc 437d
(Fig. 2A). However, CB2-transfected HEK293 cells (RBXCB2M; PerkinElmer Life Sciences) showed the strongest CB2 expression with a receptor glycosylation status comparable with the one found in HL60 cells as judged by the band size on Western blots (Fig. 2A). Based on the results from [3H]CP-55,940 displacement with anandamide (low nonspecific binding), which was used as positive control (Fig. 2B), we selected membrane preparations obtained from CB2 and CB1 overexpressing HEK293 cells, respectively, for all subsequent experiments (see Experimental Procedures). Cichoric acid, which is another prominent constituent in Echinacea, and arachidonic acid were included as negative controls in the binding studies. Anandamide showed a concentration-dependent displacement of the radioligand [3H]CP-55,940 from membrane preparations with a determined Ki of 218 149 nM (Fig. 2B). At concentrations below 100 nM, alkylamides A1 and A2 potently displaced the radioligand in a concentration-dependent manner, but the displacement curve showed a second phase at higher ligand concentrations (Fig. 2C), which may reflect solubility problems and the formation of alkylamide particles. Based on the low concentration part of the displacement curve (Fig. 2D), A1 and A2 showed high affinity toward CB2 with Ki values of 57 14 nM (A1) and 60 13 nM (A2). Significantly lower affinity was observed toward CB1 (Table 1). Cichoric acid, arachidonic acid, and the alkylamide A3 did not displace [3H]CP-55,940 (Ki 40,000 nM) from CB2 (Table 1) and therefore do not bind to the cannabinoid-binding site. Receptor ScreenTo assess the specificity of the binding to cannabinoid receptors relative to other potential targets, A1 and A2 (10 M each) were subjected to a receptor screen (see Experimental Procedures). The screen included CB1 and 49 additional pharmaceutically
FIGURE 3. Alkylamides A1 and A2 specifically bind to cannabinoid receptors. A1 and A2 (10 M each) were subjected to a receptor screen. The alkylamide A1 significantly inhibits (50%) radioligand binding to CB2 (A); the alkylamide A2 significantly inhibits (50%) radioligand binding to CB1 and CB2 (B). Data are from single measurements. In this test system, the probability that a test compound inhibits binding of radioligands by 50% by chance is 5%.
14196 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 NUMBER 20 MAY 19, 2006 Downloaded from http://www.jbc.org/ by guest on September 19, 2013
FIGURE 4. Proposed binding conformation of alkylamides A1 and A2 in the CB2 receptor. A, putative binding site for CB2 ligands is located adjacent to helices III, V, VI, and VII at the near extracellular side of the 7TM bundles. The surface is color-coded using
MAY 19, 2006 VOLUME 281 NUMBER 20 JOURNAL Downloaded from http://www.jbc.org/ by guest on September 19, 2013 OF BIOLOGICAL CHEMISTRY
14197
FIGURE 5. The effects of alkylamides, anandamide, and 2-AG on [Ca2]i in HL60 cells. The CB2 receptor surface expression on HL60-negative (1) and -positive (2) cells was determined by FACS using the antibody 3561 (representative image of three independent experiments) (A). FACS density plot of total [Ca2]i in CB2-positive HL60 cells over time was determined by fluo3/AM staining (B). 2-AG, A1, and A2 (10 M each) elevated total [Ca2]i(C). Addition of SR144528 (1 M) partially inhibited the effect. Anandamide and A3 did not significantly influence [Ca2]i. n 3 S.D. *, p 0.05; **, p 0.01; ***, p 0.001.
14198 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 NUMBER 20 MAY 19, 2006 Downloaded from http://www.jbc.org/ by guest on September 19, 2013
FIGURE 6. Overview of immunomodulatory effects of alkylamides and anandamide using different stimuli. 50 nM of drugs were incubated with CD3/PMA-stimulated, CD28/PMA-stimulated, nonstimulated, and LPS-stimulated whole blood for 18 h (see Experimental Procedures). Cytokines were quantified by FACS using CBAs. Anandamide (AN) and alkylamides from Echinacea (A1A3) differentially modulate cytokine expression (% of stimulated control). Data are mean values (n 3).
tested their sensitivity to thapsigargin. Measurements of the Ca2 sensing dye fluo3/AM were performed real time in Ca2-free buffer by FACS (Fig. 5B). Thapsigargin, which promotes the discharge of Ca2 from intracellular stores by specifically inhibiting endoplasmic reticulum Ca2-ATPase (38), led to an increase in total [Ca2]i in both cell lines (not shown). We could also confirm that 2-AG but not anandamide elevates [Ca2]i in HL60 cells, as was reported previously by Sugiura et al. (39) (Fig. 5C). 2-AG led to a significant increase (180% of vehicle control) in total [Ca2]i in CB2-positive HL60 cells, which was suppressed by the CB2 antagonist SR144528 (Fig. 5C). In these cells also A1 and A2 but not A3 significantly induced [Ca2]i elevation, and the effects could be inhibited by SR144528 (Fig. 5C). No significant modulation of [Ca2]i was detected in CB2-negative HL60 cells (not shown), which clearly suggests that CB2 is directly involved in Ca2 signaling by 2AG, A1, and A2. Effects on Constitutive Cytokine/Chemokine Expression in Human Whole Blood and HL60 CellsIn a next step we studied the effects of different concentrations of alkylamides A1, A2, and A3, anandamide, and SR144528 on constitutive cytokine/chemokine expression in human whole blood. The constitutive expression of pro-inflammatory proteins (TNF-, IL-1, and IL-6) was generally low (30 pg/ml), and only the protein level of the chemokine IL-8 (CXCL8) was typically high (100 pg/ml). First, the compounds were tested on constitutive (nonstimu-
lated) whole blood. At low concentrations (100 nM) SR144528 significantly inhibited IL-1 production, reducing constitutive expression to 75% of vehicle control (see the supplemental material). No effect on IL-1 expression was observed for any of the other compounds investigated. In contrast, the prominent constitutive expression of IL-8 was modulated by A1, A3, and anandamide (Fig. 6A). IL-8 was significantly up-regulated at low nanomolar concentrations (Fig. 6A and supplemental material). In order to test whether the modulation of IL-8 was related to interactions with the CB2 receptor in myeloid cells, we carried out experiments with CB2-positive and CB2-negative HL60 cells, both of which constitutively express IL-8 protein. Our results show that in CB2positive HL60 cells, the constitutive IL-8 expression (249 151 pg/ml) was either not affected (A1 and A3) or up-regulated (A2 and anandamide), whereas in CB2-negative HL60 cells, IL-8 expression (620 191 pg/ml) was inhibited by all compounds (see the supplemental material). Most interestingly, the CB2 antagonist SR144528 inhibited IL-8 expression in both CB2-positive and -negative HL60 cells (see the supplemental material). Compounds A1, A2, and anandamide, but not A3, significantly upregulated IL-6 protein expression to 130 160% of control levels in human whole blood (Figs. 6A and 7). Somewhat surprisingly, anandamide and A2 inhibited IL-6 at a concentration of 500 nM, thus showing a biphasic (bell-shaped) effect (Fig. 7). Because the non-CB2-binding al-
MAY 19, 2006 VOLUME 281 NUMBER 20 JOURNAL Downloaded from http://www.jbc.org/ by guest on September 19, 2013 OF BIOLOGICAL CHEMISTRY
14199
FIGURE 7. The effects of alkylamides, anandamide, and SR144528 on constitutive IL-6 expression in human whole blood. Drugs were incubated with nonstimulated whole blood for 18 h. Cytokines were quantified by CBAs. At 5 and 50 nM, all CB2 agonists (determined by binding affinity to CB2, effect on [Ca2]i, or previously published effect on cAMP) significantly increased IL-6 levels in whole blood. The up-regulation was not seen with A3. The CB2 antagonist SR144528 (1 M) significantly inhibited the effect on IL-6 (n 6 S.E., at least two different blood donors). *, p 0.05; **, p 0.01; ***, p 0.001.
FIGURE 8. Alkylamides co-stimulate CD28-activated cytokine expression and negatively regulate CD3/T-cell receptor. CD28 signals via protein kinase C (PKC) and protein-tyrosine kinases (PTKs), leading to activation of transcription factors NF-B and AP-1 (activating protein-1 (AP-1)). CD3 signals via PLC and activates inositol 3-phosphate (IP3), calcium, and diacylglycerol (DAG), leading to activation of the transcription factors nuclear factor of activated T-cells (NF-AT), NF-B, and extracellular signal-related protein kinase (Erk). Alkylamides interact with CB2 receptors and most likely another receptor, leading to an increase in total [Ca2]i (preliminary data), and activation of mitogen-activated kinases (MAPKs) (7). CD28 activation and co-stimulation with alkylamides leads to a significant super-induction (150%) of IL-3 and IL-10. On the other hand, CD3 activation and co-stimulation with alkylamides leads to a significant inhibition (20 50%) of IL-3, IL-5, and IL-10, thus suggesting that alkylamides can negatively regulate the CD3/T-cell receptor activation pathway.
14200 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 NUMBER 20 MAY 19, 2006 Downloaded from http://www.jbc.org/ by guest on September 19, 2013
FIGURE 9. Effects of alkylamides, anandamide, SR144528, and AM630 on LPS-stimulated cytokine expression in human whole blood. Drugs were incubated for 1 h prior to LPS stimulation (313 ng/ml E. coli, 055:B3 LPS) of whole blood for 18 h at 5 nM (white), 50 nM (light gray), 500 nM (dark gray), and 5000 nM (black). Cytokines were quantified by CBAs. The alkylamides A1, A2, and A3 and also anandamide and the CB2 antagonist SR144528 differentially inhibit LPS-induced cytokine expression. Only cytokines are shown where significant effects could be observed. Data from at least nine measurements derived from three different blood donors are shown (S.E.). *, p 0.05; **, p 0.01; ***, p 0.001.
MAY 19, 2006 VOLUME 281 NUMBER 20 JOURNAL Downloaded from http://www.jbc.org/ by guest on September 19, 2013 OF BIOLOGICAL CHEMISTRY
14201
CD3/PMA
pg/ml
CD28/PMA
pg/ml
p ***
PMA only
pg/ml
4266 3202 3905 3763 2695 1904 98 56 2863 1325 8571 4648 20 31 27 20 20 20
2774 533 1144 1362 722 479 84 69 113 31 5308 236 750 364 74 33 309 19 54 14 157 63
4554 469 437 403 398 567 59 47 72 48 4693 485 1324 639 55 30 392 159 41 12 61 43
* ** *
978 672 172 113 310 269 20 20 2300 951 319 205 52 20 40 13 23 8 20
amide on LPS-induced TNF- in M-enriched mononuclear cells (see the supplemental material). LPS-stimulated IL-6 was weakly inhibited at the lowest concentration (5 nM) by all compounds except for A1 (Fig. 9D). Moreover, A1 significantly up-regulated LPS-stimulated IL-8 (140%) and IL-10 (120%), but A3 and the CB2 antagonist SR144528 significantly down-regulated IL-8 (80%) (Fig. 9, E and F). In T-cell stimulation, using a combination of CD3 and CD28 as the stimulus, the resulting strong cytokine expression is derived from a combination of cellular signaling pathways (Fig. 8), and modulation of an CD3/CD28-induced response provides only limited insight into pathway specificity. Experiments with human whole blood from different donors show that CD3/PMA stimulation alone results in a distinct cytokine expression pattern from CD28/PMA stimulation (Table 2). CD28/PMA induces significantly more TNF- and GM-CSF but less IL-3 and IL-8 than CD3 (Table 2). These expression patterns were obtained reproducibly in whole blood from different blood donors (Table 2). Regarding the modulation of CD28- and CD3-stimulated cytokine expression, all three alkylamides from Echinacea, and partially also anandamide, showed a similar modulation pattern. Although CD28/PMA-stimulated cytokines (IL-3, IL-4, IL-5, and IL-10) were either not modulated (IL-4 and IL-5) or super-induced (IL-3 and IL-10), CD3/PMA-stimulated cytokines were not modulated (IL-4 and IL-10) or inhibited (IL-5 and IL-3) (Fig. 10, BD). All compounds inhibited CD3- but not CD28-induced IL-3 (produced by both TH1 and TH2 cells) (Fig. 10B). This finding suggests that protein kinase C and proteintyrosine kinase signaling mediated by CD28 stimulation of T-cells in whole blood is either directly or indirectly co-stimulated by the alkylamides and partially also by anandamide (Fig. 8). On the other hand, CD3-induced signaling via PLC is not influenced or inhibited (IL-3, IL-5, and IL-10) (Fig. 10, BD). The broad inhibitory effects by alkylamides and anandamide on LPSstimulated TNF-, IL-1, and IL12p70 (Fig. 9) was neither visible with CD3 nor CD28 stimulation (Fig. 10A). Nevertheless, A2, A3, anandamide, and SR144528 weakly but significantly inhibited TNF- expression at 50 nM by 20% (Fig. 6, C and D, and Fig. 10A).
DISCUSSION
The results presented in this study demonstrate for the first time that certain isobutylamide-type alkylamides, which are the prominent lipidlike compounds in Echinacea, bind to the human CB2 receptor with low nanomolar Ki values. The alkylamides A1 and A2 also show some affinity to the CB1 receptor (Table 1) but at 30 100 times higher concentrations (Ki 1500 nM). The total loss of binding to CB2 observed with A3 (Table 1) and the theoretical binding conforma-
tion of A1 obtained in the CB2 homology model (Fig. 4) suggest that these compounds need to adopt an extended pseudo-helical conformation for binding, as has been proposed for anandamide (43). In contrast, the docking study by McAllister et al. (44) suggests that anandamide needs to adopt a curved/U-shaped conformation to interact with the CB1 receptor. Although alkylamides are structurally similar to the endogenous cannabinoid anandamide, the anandamide molecule, containing an acyl chain with four nonconjugated double bonds, is more flexible than A1. Our current docking simulation studies indicate that alkylamides A1 and A2 fit into the putative binding pocket of the CB2 receptor (Fig. 4B) with the alkyl tail located in the hydrophobic pocket formed by the aromatic side chains of Trp-258, Phe-197, Phe-117, and Tyr-190. In this binding arrangement, the double bond between C-2 and C-4 of the hydrocarbon would be involved in a favorable - interaction with the aromatic ring of Tyr-190. Such interactions are different from those expected for anandamide, which lacks the C-2C-4 double bond. Our preliminary computer modeling studies reveal a possible CB2 binding conformation of alkylamides highlighting the importance of Tyr-190. Currently, more systematic structure-activity relationship investigations of alkylamide analogs and CB2 receptor ligand docking studies with anandamide are underway, and the results will be reported elsewhere. Based on the observation that alkylamides exhibit surfactant behavior3 and the partial lack of a concentration dependence of the effects observed in the displacement studies and biological assays, we believe that alkylamides may form micellar structures. Measurements of the critical micelle concentrations of alkylamides are in progress. A recently published study on the affinity of alkylamides from E. angustifolia to rodent cannabinoid receptors provided Ki values in the lower micromolar range (8), but in this study the displacement curve was not shown, and the solubility of alkylamides, which appears to be of key importance, was not discussed. Therefore, the discrepancy between the Ki values reported could either be due to structural differences between rodent and human CB2 receptors, experimental conditions used, or even the interpretation of displacement curves. Our results show that the CB2-binding alkylamides A1 and A2 elevate total [Ca2]i in CB2-positive but not in CB2-negative promyelocytic HL60 cells (Fig. 5). Because this effect was clearly inhibited by the CB2 antagonist SR144528, the response might be directly induced via a CB2mediated G-protein-coupled mechanism, leading to stimulation of PLC, a pathway known to be activated by CB1 agonists (45). It has been
14202 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 NUMBER 20 MAY 19, 2006 Downloaded from http://www.jbc.org/ by guest on September 19, 2013
FIGURE 10. The effects of alkylamides, anandamide, and SR144528 on CD3- and CD28-stimulated cytokine expression in human whole blood. Only those cytokines are shown for which significant effects could be observed. Drugs were incubated with stimulated (1.5 g/ml PMA; 1.0 g/ml CD3; 0.5 g/ml CD28) whole blood for 18 h with 5 nM (white bars) and 50 nM (light gray bars) of drugs. Cytokines were quantified by CBAs. Data from at least nine measurements derived from three different blood donors are shown (S.E.). *, p 0.05; **, p 0.01; ***, p 0.001.
MAY 19, 2006 VOLUME 281 NUMBER 20 JOURNAL Downloaded from http://www.jbc.org/ by guest on September 19, 2013 OF BIOLOGICAL CHEMISTRY
14203
14204 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 NUMBER 20 MAY 19, 2006 Downloaded from http://www.jbc.org/ by guest on September 19, 2013
REFERENCES
1. Goel, V., Lovlin, R., Barton, R., Lyon, M. R., Bauer, R., Lee, T. D., and Basu, T. K. (2004) J. Clin. Pharmacol. Ther. 29, 75 83 2. Barnes, J., Anderson, L. A., Gibbons, S., and Phillipson, J. D. (2005) J. Pharm. Pharmacol. 57, 929 954 3. Randolph, R. K., Gellenbeck, K., Stonebrook, K., Brovelli, E., Qian, Y., Bankaitis-Davis, D., and Cheronis, J. (2003) Exp. Biol. Med. 228, 10511056 4. Turner, R. B., Bauer, R., Woelkart, K., Hulsey, T. C., and Gangemi, J. D. (2005) N. Engl. J. Med. 353, 341348 5. Brinkeborn, R. M., Shah, D. V., and Degenring, F. H. (1999) Phytomedicine 6, 1 6 6. Percival, S. (2000) Biochem. Pharmacol. 60, 155158 7. Gertsch, J., Schoop, R., Kuenzle, U., and Suter, A. (2004) FEBS Lett. 577, 563569 8. Woelkart, K., Xu, W., Pei, Y., Makriyannis, A., Picone, R. P., and Bauer, R. (2005) Planta Med. 71, 701705 9. De Petrocellis, L., Cascio, M. G., and Di Marzo, V. (2004) Br. J. Pharmacol. 141, 765774 10. Munro, S., Thomas, K. L., and Abu Shaar, M. (1993) Nature 365, 61 65 11. Galiegue, S., Mary, S., Marchand, J., Dussossoy, D., Carriere, D., Carayon, P., Bouaboula, M., Shire, D., Le Fur, G., and Casellas, P. (1995) Eur. J. Biochem. 232, 54 61 12. Matias, I., Pochard, P., Orlando, P., Salzet, M., Pestel, J., and Di Marzo, V. (2002) Eur. J. Biochem. 269, 37713778 13. Oka, S., Yanagimoto, S., Ikeda, S., Gokoh, M., Kishimoto, S., Waku, K., Ishima, Y., and Sugiura, T. (2005) J. Biol. Chem. 280, 18488 18497 14. Quartilho, A., Mata, H. P., Ibrahim, M. M., Vanderah, T. W., Porreca, F., Makriyannis, A., and Malan, T. P., Jr. (2003) Anesthesiology 99, 955960 15. Carlisle, S. J., Marciano-Cabral, F., Staab, A., Ludwick, C., and Cabral, G. A. (2002) Int. Immunopharmacol. 2, 69 82 16. Parolaro, D., Massi, P., Rubino, T., and Monti, E. (2002) Chem. Phys. Lipids 108, 169 190 17. Klein, T. W. (2005) Nat. Rev. Immunol. 5, 400 411 18. Klein, T. W., Newton, C., Larsen, K., Lu, L., Perkins, I., Nong, L., and Friedman, H. (2003) J. Leukocyte Biol. 74, 486 496 19. Croxford, J. L., and Yamamura, T. (2005) J. Neuroimmunol. 166, 318 20. Sarfaraz, S., Afaq, F., Adhami, V. M., and Mukhtar, H. (2005) Cancer Res. 65, 16351641 21. Blazquez, C., Casanova, M. L., Planas, A., Del Pulgar, T. G., Villanueva, C., FernandezAcenero, M. J., Aragones, J., Huffman, J. W., Jorcano, J. L., and Guzman, M. (2003) FASEB J. 17, 529 531 22. Steffens, S., Veillard, N. R., Arnaud, C., Pelli, G., Burger, F., Staub, C., Karsak, M., Zimmer, A., Frossard, J. L., and Mach, F. (2005) Nature 434, 782786 23. Devane, W. A., Dysarz, F. A., III, Johnson, M. R., Melvin, L. S., and Howlett, A. C. (1988) Mol. Pharmacol. 34, 605 613 24. Kaminski, N. E., Abood, M. E., Kessler, F. K., Martin, B. R., and Schatz, A. R. (1992) Mol. Pharmacol. 42, 736 742 25. Xie, X. Q., Chen, J. Z., and Billings, E. M. (2003) Proteins 53, 307319 26. Woelkart, K., Koidl, C., Grisold, A., Gangemi, J. D., Turner, R. B., Marth, E., and Bauer, R. (2005) J. Clin. Pharmacol. 45, 683 689 27. Matthias, A., Addison, R. S., Penman, K. G., Dickinson, R. G., Bone, K. M., and Lehmann, R. P. (2005) Life Sci. 77, 2018 2029 28. Calignano, A., La Rana, G., Giuffrida, A., and Piomelli, D. (1998) Nature 394, 277281 29. Cheng, Y. C., and Prusoff, W. H. (1973) Biochem. Pharmacol. 22, 3099 3108 30. Palczewski, K., Kumasaka, T., Hori, T., Behnke, C. A., Motoshima, H., Fox, B. A., Le Trong, I., Teller, D. C., Okada, T., Stenkamp, R. E., Yamamoto, M., and Miyano, M. (2000) Science 289, 739 745 31. Bauer, R., Reminger, P., and Wagner, H. (1988) Phytochemistry 27, 2339 2342 32. Raitio, K. H., Salo, O. M., Nevalainen, T., Poso, A., and Jarvinen, T. (2005) Curr. Med.
MAY 19, 2006 VOLUME 281 NUMBER 20 JOURNAL Downloaded from http://www.jbc.org/ by guest on September 19, 2013 OF BIOLOGICAL CHEMISTRY
14205
14206 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 NUMBER 20 MAY 19, 2006 Downloaded from http://www.jbc.org/ by guest on September 19, 2013